DNA methylation is an important epigenetic mechanism having several regulatory functions such as X-chromosome inactivation, genomic imprinting, and suppression of retro-transposition (Goll and Bestor 2005). This process of methylation involves the transfer of a methyl group from S-adenosyl L-methionine (AdoMet) to the C5 carbon of a cytosine (C) by an enzyme called DNA methyltransferase (Mtases) to produce 5-methylcytosine (5mC) (Goll and Bestor, 2005). The human genome contains ˜28 million CpG sites, about 70% of which are methylated at the 5 position of the cytosine (Edwards at al., 2010). Aberrant or abnormal DNA methylation has been linked to a growing number of human diseases including Immunodeficiency, Centromere instability and Facial anomalies (ICF) syndrome, fragile X syndrome, and other developmental diseases, age-related neurodegenerative disorders, diabetes and cancer (Robertson et al., 2005; reviewed by Goll and Bestor, 2005). Hence, epigenetic changes in DNA methylation status are increasingly being studied for their role in both normal and disease-associated phenotypic changes, including the Roadmap Epigenomics Project launched by NIH to create reference epigenomes for a variety of cell types. To fulfill this goal, genome-wide methods and techniques that can comprehensively profile DNA methylation status with single base resolution and high throughput are essential (Suzuki et al., 2008, Laird, 2010).
Over 30 methylation analysis technologies have been developed, but all have shortcomings (Laird, 2010). Bisulfite genomic sequencing (BGS), reported by Susan Clark and Marianne Frommer in 1994 (Clark et al., 1994), is regarded as the best available method. However, BGS has several serious shortcomings: (1) there is a severe loss of sequence information upon bisulfite conversion which produces sequences that cannot be aligned to the genome; (2) strong biases against GC-rich sequences (Edwards et al., 2010); (3) bisulfite conversion artifacts, and (4) the need for large amounts of long starting DNA due to high rates of strand cleavage under the harsh reaction conditions (Warnecke et al., 2002, 1997). In spite of the development of over 30 technologies that enable identification of the methylation status of DNA (methylome analysis), the determination of whole-genome methylation patterns remains difficult and expensive (Clark et al., 1994). Major improvements or new approaches overcoming the above mentioned limitations are needed to further advance genome-wide DNA methylation profiling.